Molecular Cancer Therapeutics

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging cancers to treat due to the dismal survival rate, poor post-treatment outcome and profound resistance to a wide range of therapies. With mutant KRAS being a key driver, small molecule inhibitors targeting KRAS or pan-RAS (KRASi) have demonstrated exciting preclinical and early clinical anti-tumor efficacy, and the pan-RAS(ON) inhibitor daraxonrasib (RMC-6236) recently achieved Phase 3 clinically meaningful improvements in patient survival compared to chemotherapy. But resistance to RAS/KRAS inhibitor inevitably develops, which limits and compromises the treatment outcome. In this study, we investigated the combination of stereotactic body radiation therapy (SBRT) and KRAS inhibition (MRTX1133 and daraxonrasib) in the treatment of preclinical PDAC models. We found that this combination strategy synergistically suppresses PDAC cell growth in vitro and enhances tumor control while minimizing local recurrence in orthotopically implanted KPC (LSL-KrasG12D/+;Trp53R172H/+;Pdx1-Cre) murine PDAC tumors in vivo. As radiation therapy (RT) induces ferroptosis in multiple cancer types and mutant KRAS promotes various anti-ferroptotic mechanisms, we tested the role of ferroptosis in promoting tumor-control efficacy. Intriguingly, the addition of a ferroptosis inhibitor, liproxstatin-1, to the combination therapy significantly abrogated the in vivo synergism between SBRT and KRAS inhibition, suggesting that treatment-induced ferroptosis at least partially drives the synergistic efficacy of this combination strategy. Our study indicates that this SBRT-KRASi combination has the potential to overcome treatment resistance and improve outcomes in PDAC patients. These data directly support the design of a planned multi-center Phase 2 clinical trial with this combination strategy in locally advanced PDAC.

PurposeCisplatin (CDDP) is the most active chemotherapy for ovarian cancer; primary or acquired resistance signals a poor prognosis. Nelfinavir (NFV), an HIV protease inhibitor, has demonstrated anti-tumor activity in multiple cancer models, but its interaction with CDDP in ovarian cancer has yet to be demonstrated. In this work, we addressed whether the combination of CDDP and NFV provides treatment advantage in platinum (Pt)-resistant ovarian cancer cells. MethodsDrug synergy between NFV and CDDP was assessed using cell vitality assays and Loewe additivity modelling. Apoptotic and pyroptotic signalling were evaluated by immunoblotting, mitochondrial membrane potential analysis, and lactate dehydrogenase (LDH) release, and caspase inhibition. Transcriptomic changes were assessed by bulk mRNA sequencing followed by differential gene expression analysis and gene set enrichment analysis. ResultsNFV synergized with CDDP to reduce the viability of Pt-resistant ovarian cancer cells, promoting a regulated lytic cell death phenotype involving apoptotic and pyroptotic features. Combination treatment induced caspase-8 and caspase-3 activation, and downstream gasdermin E (GSDME) processing. Inhibition of caspase-3 significantly attenuated cell death, and caspase-8 inhibition rescued viability and prevented Bid cleavage, caspase-3 activation, and GSDME cleavage. These effects occurred in the context of enhanced endoplasmic reticulum stress, increased DNA damage with reduced DNA repair, and impaired Akt-driven survival signalling. ConclusionsOur findings establish that NFV synergizes with CDDP in killing Pt-resistant ovarian cancer cells by promoting a caspase-8-dependent apoptotic-to-secondary pyroptotic response, supporting further investigation of NFV as a potential drug to be repurposed to increase the efficacy of Pt-based therapy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by a dense desmoplastic tumor microenvironment (TME) that limits drug penetration and promotes immune evasion. Effective treatment, therefore, requires simultaneous modulation of multiple signaling pathways. Here, we describe a directed polypharmacological strategy to identify dual modulators of c-MET and Smoothened (SMO), aiming to disrupt the protective stroma through SMO inhibition while directly suppressing tumor cell survival via c-MET targeting. An AI-guided virtual screening workflow combining the machine-learning platform PyRMD, trained on known c-MET and SMO ligands, with structure-based molecular docking was applied to a library of over 9 million compounds. This approach led to the identification of compound 21, an aminopyrimidine-benzamide-phenoxyquinoline derivative, as a dual c-MET/SMO inhibitor. Biochemical and cellular studies demonstrated that compound 21 selectively binds the SMO orthosteric site (pKi = 5.60), inhibits agonist-induced GLI (Glioma-associated oncogene) signaling (pIC50 = 5.50), and potently suppresses c-MET kinase activity (pIC50 = 6.94). Western blot analyses further revealed that compound 21 promotes ubiquitin-proteasome-mediated degradation of c-MET, eliminating receptor availability and limiting compensatory resistance signaling. In 3D heterotypic models comprising MIAPaCa2 pancreatic cancer cells and CAF154-hTERT fibroblasts, dual inhibition of SMO-mediated stromal support and c-MET-driven tumor progression resulted in greater cytotoxicity than the combination of the selective inhibitors Sonidegib and PHA-665752. Overall, compound 21 overcomes stromal-mediated resistance, enhances tumor cell death, and validates dual SMO/c-MET targeting as a promising single-agent therapeutic strategy for PDAC. One Sentence SummaryAn AI-identified dual SMO/c-MET inhibitor overcomes stromal resistance and degrades c-MET to suppress pancreatic cancer.

177Lu-PSMA-617 (PluvictoTM, Lu-177 RLT) is an FDA-approved targeted radioligand therapy (RLT) for metastatic castration-resistant prostate cancer (mCRPC), but its durability of response to this singular approach poses a challenge to the field. Chimeric antigen receptor (CAR) T cell therapy has revolutionized clinical practice for hematological malignancies, but its clinical development for solid tumors, including mCRPC, has been encumbered by antigen heterogeneity and the immunosuppressive tumor microenvironment (TME). Here, we evaluate the therapeutic combination of Lu-177 RLT and PSCA-CAR T cells to overcome these barriers. In human xenograft and mouse syngeneic prostate cancer models with homogeneous or heterogeneous antigen expression, the sequential administration of Lu-177 RLT, cyclophosphamide (Cy), and PSCA-CAR T cells improves tumor control and prolongs survival compared to monotherapies. Mechanistically, Lu-177 RLT alone or with Cy remodels the TME by promoting pro-inflammatory myeloid responses and activating endogenous T cells, while enhancing CAR T cell activation and effector function. We additionally evaluated 225Ac-PSMA-617 RLT as an emerging approach in combination with CAR T cells and observed anti-tumor responses, supporting its potential as an alternative RLT partner. These findings support RLT as an immune priming strategy to enhance CAR T cell therapy and provide a rationale for clinical translation of this combination in mCRPC. One Sentence SummaryCombining 177Lu-PSMA-617 radioligand therapy with PSCA-CAR T cells improves tumor control and survival in prostate cancer models by overcoming the antigen heterogeneity and reshaping the immunosuppressive tumor microenvironment.

Colorectal cancer (CRC) remains a significant contributor to cancer-associated deaths worldwide, indicating the need for new therapeutic targets and modalities. Antibody-drug conjugates (ADCs) have demonstrated remarkable potential for the treatment of various cancer types, although their efficacy as monotherapies is often limited by insufficient targeting of tumor heterogeneity, dose-limiting toxicities, and drug resistance. Accordingly, multi-targeting therapeutic strategies, such as bispecific ADCs (bsADCs), which simultaneously target two cancer-associated antigens or non-overlapping epitopes on the same antigen, may prove more effective at overcoming resistance and eliminating tumors compared to monospecific ADCs. In this work, we describe the development of EGFR:LGR5 bispecific antibodies (bsAbs) and bsADCs. EGFR:LGR5 bsAbs were shown to internalize to the lysosome to a greater extent than EGFR- and LGR5-targeting monoclonal antibodies (mAbs) and drive EGFR lysosomal degradation in an LGR5-mediated fashion. However, EGFR:LGR5 bsAbs exerted suboptimal cytotoxicity in CRC cell lines. We therefore engineered an EGFR:LGR5 bsADC that demonstrated 100- to 1000-fold enhanced efficacy over a previously developed LGR5-targeting monospecific ADC (8E11-CPT2) with an identical linker-payload in CRC cell lines of various genetic backgrounds and EGFR and LGR5 expression levels. EGFR:LGR5 bsADC potency was strongly correlated with cell line sensitivity to the CPT2 payload. EGFR:LGR5 bsADC induced tumor regression in select RASMUT CRC xenograft models and demonstrated superior antitumor activity and prolonged survival benefit in all evaluated models versus EGFR mAb cetuximab (CTX), bsAb, and 8E11-CPT2. These findings strongly support the further development of EGFR and LGR5 dual-targeting approaches for CRC and other EGFR- and LGR5-expressing malignancies. One Sentence SummaryEGFR:LGR5 bsADCs exert robust antitumor activity and outperform EGFR:LGR5 bsAb and LGR5 monospecific ADC in RASWT and RASMUT colorectal cancer models.

Background: Medulloblastoma (MB) is the most common malignant brain tumour in children, and aggressive subgroups are frequently driven by the oncoproteins MYC or MYCN. Direct therapeutic targeting of MYC/MYCN has been challenging because of their intrinsically disordered protein structures. The aim of this study was to determine whether novel SE486-11 analogues (UNSW-SCs) can therapeutically target MYC/MYCN-driven MB. Methods: The anticancer activity of UNSW-SCs was assessed in MB cell lines with differential MYC/MYCN expression. Target engagement was evaluated using surface plasmon resonance and drug affinity responsive target stability assays. Blood-brain barrier penetration, MYC/MYCN protein degradation, cell cycle effects, apoptosis, DNA damage, and synergy with histone deacetylase (HDAC) inhibitors were examined. Therapeutic efficacy was evaluated in murine models of MYC- and MYCN-driven human MB. Results: UNSW-SCs showed potent anticancer activity, with preferential selectivity toward MB cells expressing high MYC/MYCN levels and IC50 values ranging from 0.22 to 1.18 M. The lead molecule, UNSW-SC-22, directly bound MYC, crossed the blood-brain barrier, and achieved a brain-to-plasma ratio of 1.44 at peak concentrations. UNSW-SC-22 induced MYC/MYCN-dependent cytotoxicity associated with enhanced proteasomal degradation, cell cycle arrest, apoptosis, and DNA damage. Combined treatment with HDAC inhibitors further reduced MYC/MYCN protein levels, increased DNA damage, and enhanced apoptosis. In vivo, UNSW-SC-22, either alone or with entinostat, significantly suppressed intracranial tumour growth and prolonged survival. Conclusions: UNSW-SC-22 is a brain-penetrant MYC/MYCN-targeting molecule with potent preclinical activity in MYC/MYCN-driven MB, supporting its development as a monotherapy or combination strategy with HDAC inhibition.

Homologous recombination deficiency (HRD) is an actionable vulnerability found in a substantial fraction of human cancers, yet current HRD-directed therapies are limited by toxicity, incomplete responses, and acquired resistance. Many DNA-damaging agents were developed before DNA repair biomarkers were available, suggesting that abandoned agents may harbor previously unrecognized genotype-selective activity. Here, through a focused screen of DNA-damaging agents in isogenic homologous recombination-proficient and -deficient models, we identify CB1954, a decades-old nitrobenzamide aziridine prodrug, as highly selective for BRCA2-deficient tumor cells. CB1954 forms DNA interstrand crosslinks independent of HR status, but selectively induces DNA-damage signaling, apoptosis, and loss of clonogenic survival in HR-deficient cells. Targeted DDR CRISPR screening and isogenic validation define a distinct repair dependency for the Fanconi anemia and homologous recombination pathways, with limited dependence on mismatch repair or nucleotide excision repair. Genetic and pharmacologic perturbation of NQO2, the bioactivating enzyme for CB1954, reveals a bifurcated mechanism in which NQO2-dependent activation selectively contributes to HRD cytotoxicity, while aziridine-dependent lesions likely account for residual activity in HR-proficient cells. CB1954 exhibits favorable preclinical pharmacokinetic properties and genotype-dependent antitumor activity in BRCA2-deficient xenografts. These findings reposition CB1954 as a historically overlooked HRD-selective agent and demonstrate that biomarker-guided profiling of DNA-damaging agents can uncover new opportunities for precision oncology.

Background: Ovarian cancer (OC) exhibits substantial heterogeneity in response to platinum-based chemotherapy, resulting in variable clinical outcomes and frequent recurrence. Current biomarkers, including serum CA-125 kinetics and BRCA mutational status, incompletely predict therapeutic response. We investigated whether patient-derived organoids (PDOs) could functionally stratify chemotherapy sensitivity and better reflect patient-specific clinical behaviour. Methods: Twenty patients with OC treated between January 2024 and May 2026 were included, from whom fourteen PDO lines were successfully established. Eight PDOs with robust low-passage expansion and comprehensive longitudinal follow-up underwent functional profiling against carboplatin, paclitaxel, olaparib, and doxorubicin. Drug responses were assessed using half-maximal inhibitory concentration (IC50) and area under the curve (AUC) analyses and integrated with radiological response, serum CA-125 kinetics, BRCA status, and progression-free survival (PFS). Results: Clinical outcomes varied considerably despite similar platinum-taxane regimens. Although post-treatment CA-125 reduction was associated with prolonged PFS, neither CA-125 kinetics nor BRCA mutational status consistently predicted therapeutic response. PDO-guided functional stratification segregated tumours into four clinically relevant platinum-taxane response phenotypes: dual-sensitive, platinum-sensitive/taxane-resistant, platinum-resistant/taxane-sensitive, and dual-resistant. These functional categories closely mirrored radiological response, CA-125 normalisation, and disease progression patterns. PDOs exhibiting low IC50 and AUC values were associated with durable clinical benefit, whereas resistant PDOs tracked with persistent disease and early recurrence. Conclusions: PDO-guided functional stratification captures clinically meaningful therapeutic heterogeneity in OC and complements conventional biomarkers by directly measuring tumour-specific drug susceptibility. Prospective integration of PDO testing may facilitate patient-specific therapeutic selection and support functional precision oncology approaches in ovarian cancer.

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer.

Androgen receptor (AR) antagonists, such as enzalutamide, suppress prostate cancer (PCa) cells to achieve temporary therapeutic effects. In addition to tumor cell-autonomous suppressive function, AR antagonists can also potentially exert anti-tumor effects via mitigating cytotoxic T cells exhaustion. However, strategies for effectively harnessing enzalutamides immunotherapeutic effects remain elusive. In studying a recently described glutamine antagonist prodrug (DRP-104) in PCa models, we found that despite the initial response, tumors ultimately became resistant. Intriguingly, compared to the untreated (DRP-104 treatment-naive) tumors, the resistant tumors became highly susceptible to enzalutamide in vivo. Additionally, treating tumors with DRP-104 and enzalutamide simultaneously also yielded superior therapeutic efficacy. We demonstrated that DRP-104 therapy promoted the infiltration of CD8+ T cells as well as regulatory T cells (Treg) in responsive tumors, and the tumor-infiltrating Treg were mostly depleted upon enzalutamide treatments. Mechanistically, we showed that Treg differentiation from mouse CD4+ T cells was attenuated by enzalutamide. We further demonstrated that Treg induction was accompanied by the interaction between AR and aryl hydrocarbon receptor (AhR), the nuclear receptor indispensable for Treg differentiation, in the nuclei of CD4+ T cells, and this interaction was diminished by enzalutamide. In further support of AR signaling in Treg biogenesis, analysis of available gene expression datasets found that AR expression was elevated in Treg when compared to CD4+ T cells in human peripheral blood mononuclear cells (PBMCs). In addition, it was positively correlated with Treg module scores in several human cancer types. Finally, using an anti-GPC3 (Glypican 3) vaccination model, we demonstrated that CD4+ T cells subjected to Treg induction in the presence of enzalutamide were less effective in protecting GPC3-expressing tumor cells from CD8+ T cells cytotoxic killing. Collectively, these results suggest that AR promotes Treg s differentiation and/or immunosuppressive functions, and nominate enzalutamide as a Treg-mitigating agent for potentiating immunotherapies. Our results also demonstrate that an otherwise unintended, Treg-promoting property of DRP-104 can be leveraged to unleash the immune-regulatory function of enzalutamide for the treatment of PCa.

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5{beta}-dihydrotestosterone (5{beta}-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5{beta}-DHT and related 5{beta}-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5{beta}-DHT and 3{beta}-etiocholanediol (3{beta}-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2, with weaker activity than testosterone, whereas other 5{beta} metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5{beta}-DHT and 3{beta}-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5{beta}-DHT and 3{beta}-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5{beta}-DHT, but not 3{beta}-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5{beta}-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects. HighlightsO_LI5{beta}-DHT and 3{beta}-ecdiol promote AR-dependent prostate cancer cell growth C_LIO_LIBoth are weaker AR agonists than testosterone by RNA-seq and qPCR C_LIO_LISupraphysiologic 5{beta}-DHT suppresses growth via AR-mediated senescence C_LIO_LIGrowth suppression extends to AR mutants W742C and H875Y C_LIO_LI5{beta}-DHT may be a lower-androgenicity testosterone surrogate for BAT C_LI

Patient-derived organoids (PDOs) have transformed translational cancer research, allowing tractable models that better represent clinical features than traditional immortalized cell lines. Here we describe two PDOs with differential responses to carboplatin derived from sequential ascites fluid collections from a patient with high-grade mullerian carcinoma, that could not be further subclassified on the omental biopsy. Uterine origin was clinically excluded by pelvic imaging/CT scan of the uterus and absence of vaginal bleeding. Successful derivation from independent collections enabled comparison of intra-patient heterogeneity across sequential ascites samples and demonstrates that PDO efficiency rate is at least partly patient-specific or tumor-dependent. We performed long-read whole genome sequencing on the two PDOs, OC104 and OC109, to better characterize the structural variant landscape while also obtaining information on single nucleotide variants and DNA methylation. In addition to confirming single nucleotide variants noted in clinical sequencing (TP53, KRAS, SPOP, PPP2R1A, KMT2D), we identified additional variants in TSC2, NCOR2, and CTNNA2 that are predicted to be likely pathogenic. The spectrum of mutations, particularly the coincident KRAS and TP53, highlighted unexpected overlap with ovarian mucinous carcinoma. We also identified larger insertions and deletions that result in non-synonymous variants in MUC5AC, TPRX1, and BMX, as well as four translocation events, including two that could not have been resolved with short-read sequencing. Differentially methylated promoters between the two PDOs include 201 oncogenes and tumor suppressor genes, with HNF1A, MSI2, and SETBP1 having methylation directions consistent with these genes' roles in platinum response differences observed between the PDOs. Notably, the clonal nature of PDOs produced from two samples taken one week apart is important for the field to appreciate, particularly since they have clonal differences in platinum response. The temporal differences in clonality may indicate a limitation of low volume sampling, however may provide opportunity to longitudinally predict clinical outcomes. We also demonstrate the ability of long-read sequencing to add detail into the genomics and epigenetics of ovarian cancer.

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Genetic uniqueness of the tumor microenvironment significantly influences cancer growth, survival, and response to therapy, independent of the cancer cells intrinsic properties or the adaptive immune system. Using genetically distinct Rag1-/- mouse models, this study shows that different strains exhibit varied tumor growth kinetics and survival outcomes when xenografted with identical leukemic and solid tumor cell lines. This study further highlights the critical role of the myeloid immune compartment and shows that disrupting both lymphoid and myeloid systems alters cancer progression. These results also reveal that the tumor microenvironment can permanently alter cancer cell phenotypes and significantly affect chemotherapy efficacy, as seen with Cisplatins varying effects across strains. These findings underscore the importance of considering genetic background in preclinical cancer models, suggesting that reliance upon a single mouse strain may lead to incomplete conclusions about cancer biology and treatment efficacy. SUMMARY STATEMENTPre-clinical xenograft mammalian models are used to study human diseases. Here we report that the genetic uniqueness of the tumor microenvironment, independent of the immune system, can determine the fate of cancer progression, survival, and therapy response.

Clinical-grade RAS inhibitors raise an unresolved question as to whether KRAS-alleles impose constraints on adaptive resistance that can be exploited therapeutically. Using daraxonrasib (RMC-6236), a multi-selective RAS(ON) inhibitor, we compared resistance mechanisms between KRASG12D and KRASG12R, alleles with fundamentally different RAS network dynamics. Daraxonrasib inhibited KRASMUT primarily through steric occlusion of effector binding, while engaging RASWT only modestly ([~]20%). KRASG12R is marked by its inability to transactivate RASWT, and it was observed that daraxonrasib resistant KRASG12R PDAC cells utilize EGFR/RASWT-GTP signaling as the dominant adaptive route. In contrast, KRASG12D resistance arose through retained KRASG12D-GTP signaling, with a decrease of cyclophilin A (CypA) protein, the binding partner required for daraxonrasib activity. The shift from KRASG12R dependence to the EGFR/RASWT conferred sensitivity to trametinib. We confirmed this clinically: a KRASG12R PDAC patient who progressed after 10 months on daraxonrasib showed intratumoral EGFR/RASWT activation, and rapid 3D-bioprinted patient-derived toroid modeling predicted sensitivity to trametinib-based combination therapy. Given the aggressive disease trajectory and lack of response to the two immediately preceding lines of therapy, sixth-line trametinib-based combination therapy achieved approximately 5 months of disease control. This patient ultimately achieved 40 months of overall survival, far exceeding the 8-12 month median for metastatic PDAC. Collectively, these data establish a framework in which allele-specific RAS network topology dictates the adaptive resistance landscape, enabling rational selection of targeted therapies with meaningful clinical benefit in metastatic PDAC. STATEMENT OF SIGNIFICANCEDaraxonrasib resistance mechanisms have allele-specific routes: CypA becomes downregulated in KRASG12D and reliance on EGFR/RASWT in KRASG12R. Rapid patient-derived toroids identified sixth-line targeted therapy strategies with an overall survival of 40 months.

Canine hemangiosarcoma (HSA) is an aggressive endothelial malignancy with limited therapeutic options, and its progression is closely associated with vascular architecture, stromal remodeling, and inflammatory cell recruitment. Sulfoquinovosylacylpropanediol (SQAP) is a sulfoquinovosyl lipid radiosensitizer reported to affect angiogenic and tumor-microenvironmental pathways, but its effects in canine HSA are unknown. Here, we evaluated SQAP in canine HSA cell lines and patient-derived xenograft (PDX) models. SQAP showed minimal direct cytotoxicity against HSA cell lines in vitro, whereas it significantly suppressed tumor growth in three canine HSA PDX models. Transcriptome analysis of SQAP-treated HSA PDX tumors detected more SQAP-responsive genes in mouse host-derived cells than in canine tumor cells. Gene-set enrichment analysis of the mouse host-derived fraction showed positive enrichment of angiogenesis, hypoxia, and stromal remodeling-related gene sets after SQAP treatment. Subsequent tissue analysis showed that SQAP reduced host-derived CD31-positive vascular area and increased -smooth muscle actin coverage of remaining vessels in two of the three PDX models, while altering macrophage-associated marker profiles in a model-dependent manner. These findings indicate that SQAP suppresses canine HSA PDX growth primarily through vascular and macrophage-associated remodeling of the tumor microenvironment rather than direct tumor-cell cytotoxicity.

IntroductionThe RAS(ON) multi-selective daraxonrasib (RMC-6236) inhibitor is effective in patients with NSCLC KRAS mutant cancers. Tolerance to daraxonrasib invariably develops by mechanisms that remain unclear. There is no known involvement of the M1C oncogenic protein in daraxonrasib resistance. MethodsNSCLC H358 KRAS(G12C), H2122 KRAS(G12C) and patient derived MGH1112 KRAS(G12C) cells with acquired daraxonrasib resistance were investigated for M1C dependence in studies of SHP2, STAT1/3 and NF-KB activation, clonogenicity, and self-renewal capacity. ResultsWe demonstrate that M1C is induced as a protective response in NSCLC KRAS(G12C) mutant cells treated with daraxonrasib. We report that M1C forms novel cell membrane-associated biomolecular condensates with the SHP2 protein tyrosine phosphatase in driving daraxonrasib resistance. M1C integrates SHP2 activation with induction of (i) oncostatin-m/gp130/STAT3 signaling, and (ii) the NF-{kappa}B-mediated epithelial-mesenchymal transition (EMT) pathway. The functional significance of this M1C-driven pathway is supported by the demonstration that targeting STAT3 and NF-{kappa}B reverses daraxonrasib resistance. Consistent with M1C dependence, we also show that targeting M1C is effective against daraxonrasib-resistant NSCLC KRAS mutant cell line and tumor models. In contrast, M1C drives sotorasib resistance by STAT1-mediated inflammatory signaling, demonstrating that M1C confers resistance to KRAS(G12C)-selective and RAS(ON) tri-complex inhibitors by noncongruent mechanisms. ConclusionsThese findings demonstrate that M1C is required for daraxonrasib tolerance and is a potential target for the treatment of patients with NSCLC KRAS(G12C) mutant tumors refractory to this agent.

IntroductionNectin-4 targeting antibody-drug conjugate (ADC) enfortumab vedotin (EV), in combination with pembrolizumab, is the first-line treatment for patients with locally advanced or metastatic urothelial carcinoma (UC). Optimal treatment strategies for patients who are non-responders or progress on EV with pembrolizumab remain an unmet clinical need. We sought to characterize ADC and immunotherapy (IO)-associated target expression profiles to identify candidate therapeutic vulnerabilities beyond EV. MethodsWe conducted a literature review to identify ADC and IO targets with approved or investigational relevance in UC. Unsupervised hierarchical clustering was used to identify clusters of target gene expression in RNA-seq data. Transcriptomic clustering analyses were performed in 434 patients from The Cancer Genome Atlas Bladder Urothelial Carcinoma cohort (TCGA-BLCA) and validated in an independent cohort of 478 patients from the Oncology Research Information Exchange Network (ORIEN) consortium. Proteomic interrogation of these targets was performed using mass spectrometry data from additional cohort of 116 patients. Differential gene expression analyses evaluated associations between target expression patterns, histologic variants, and consensus molecular subtypes of muscle-invasive bladder cancer (CMIBC). ResultsWe identified 13 ADC and 10 IO-associated targets with translational relevance in UC. Transcriptomic analyses revealed three reproducible clusters of overexpressed target genes across independent cohorts: 1) a luminal/epithelial-associated cluster enriched for VTCN1, SLITRK6, FGFR3, NECTIN4, TACSTD2, ERBB2, and ERBB3; 2) an immune target predominant cluster enriched for BTLA, LAG3, PDCD1, TIGIT, CTLA4, TNFRSF9, TNFRSF18, TNFRSF4; and 3) a basal/neuroendocrine-associated cluster characterized by CD274, F3, NT5E, EGFR, MET and DLL3. Similar clusters were largely conserved at the proteomic level. Adenocarcinomas overexpressed ERBB3 compared to neuroendocrine and squamous cell carcinomas. Pure squamous cell carcinomas overexpressed TACSTD2 compared to adenocarcinomas. In CMIBC subtypes, basal/squamous tumors expressed higher levels of CD274, EGFR, F3, LAG3, NT5E, and TNFRSF18, whereas luminal tumors demonstrated higher ERBB2 and ERBB3 expression. Neuroendocrine-like tumors showed higher DLL3 expression compared to all other subtypes. Tumors with low expression of NECTIN4, TACSTD2, and FGFR3 were enriched for alternative targets including DLL3, CD274, and CD276. Our findings provide a framework for hypothesis-driven therapeutic prioritization in advanced UC. Conclusions: UC is characterized by reproducible, biologically distinct patterns of ADC and IO target expressions. The degree of expression of NECTIN4 was positively associated with TACSTD2, FGFR3 and inversely associated with DLL3, CD276, and CD274, supporting alternative biologically informed treatment strategies besides EV . Histologic variants and molecular subtypes of UC also display distinct patterns of target expression. This study provides the first integrated transcriptomic framework linking ADC and IO target co-expression patterns for hypothesis-driven therapeutic prioritization. These findings provide a basis for rational ADC and immunotherapy development in advanced UC and support prospective proteomic validation in treatment stratified cohorts. Statement of Translational RelevanceEnfortumab vedotin plus pembrolizumab has redefined first-line therapy for advanced urothelial carcinoma, yet treatment selection following resistance or progression remains undefined. In this study, we integrate transcriptomic and proteomic analyses across independent cohorts to define reproducible patterns of antibody-drug conjugate (ADC) and immunotherapy target co-expression in urothelial carcinoma. We identify biologically distinct target-expression patterns that are associated with histologic and molecular subtypes and demonstrate coordinated and, in some cases, mutually exclusive relationships among therapeutically actionable targets. These findings have direct translational implications. First, they provide biologic rationale for rational sequencing and combination strategies based on co-expressed targets in NECTIN4-enriched tumors. Second, they identify alternative therapeutic vulnerabilities, including DLL3- and CD274-associated pathways, in tumors with low NECTIN4 expression, a population potentially enriched for resistance to EV-based therapy. Finally, this framework establishes a foundation for biomarker-driven clinical trials in urothelial carcinoma and supports the development of precision therapeutic approaches beyond current standards.

Targeting oxidative phosphorylation (OXPHOS) represents an attractive therapeutic strategy in acute myeloid leukemia, which exhibits exceptional dependence on mitochondrial respiration compared to normal hematopoietic cells. However, clinical attempts to exploit this vulnerability have been limited by on-target toxicity to healthy tissue. Here, we comprehensively compare the cellular consequences of inhibiting distinct nodes of the electron transport chain in AML. We demonstrate that selective inhibition of the F1 subunit of ATP synthase with EB2023 (ammocidin A) delivers an energetic stress to AML cells without the profound redox stress that characterizes complex I inhibition, preventing NAD/NADH imbalance and allowing continued TCA cycling. Further, the duration of OXPHOS inhibition is transient in nature in vivo, a finding revealed through pharmacokinetic and serial pharmacodynamic monitoring of AMPK phosphorylation accompanied by OPA1-mediated mitochondrial structural remodeling that primes AML cells for BCL2 inhibitor synergy. EB2023 in combination with venetoclax demonstrates potent anti-AML activity across cell lines and patient-derived xenograft models at doses that spare normal hematopoietic progenitors and avoid the neuropathy and sustained detrimental systemic metabolic rewiring in healthy tissues associated with prior efforts to target OXPHOS. These findings establish F1-selective ATP synthase inhibition as a clinically actionable therapeutic strategy in AML and establish the duration of OXPHOS inhibition as a critical and previously underappreciated determinant of therapeutic index.

Peritoneal carcinomatosis is a major cause of death in gastric cancer, yet effective therapies remain limited. Tumor-derived soluble factors are increasingly recognized as key regulators of the peritumoral microenvironment. Here, we nominate osteopontin (SPP1) as a tumor-derived mediator that orchestrates macrophage-driven immunoregulation in gastric peritoneal carcinomatosis. Using integrated analyses of human clinical datasets and murine models, we demonstrate that tumor-secreted SPP1 promotes macrophage recruitment and induces tolerogenic IL-10 production. Clinically, SPP1 correlated with inferior overall survival and progression-free survival in gastric cancer. In syngeneic murine models of gastric peritoneal carcinomatosis, intracavitary pharmacologic inhibition of SPP1 restricted peritoneal dissemination, impaired macrophage infiltration and suppressed IL-10 production. Consistent with these findings, macrophage depletion phenocopied antitumor effects of SPP1 inhibition, resulting in decreased metastatic burden. Collectively, these findings define a mechanism of tumor-macrophage crosstalk that promotes peritoneal dissemination and provide a rationale for therapeutic targeting of SPP1 in gastric peritoneal carcinomatosis.